Project Summary/Abstract Recently ribosome subpopulations that differ in their composition, lacking individual ribosomal proteins (RPs), or containing specific modifications have garnered a lot of interest. In the case of RP content, multiple studies have shown that ribosomes lacking specific RPs are present in cells, including ribosomes deficient in Rps26. While their physiological relevance remains unclear in most cases, the Karbstein lab has recently demonstrated that ribosomes lacking Rps26 are produced specifically under high salt and pH stress to enable the preferential translation of mRNAs encoding proteins from the Hog1 and Rim101 pathways that are required for the response to these stresses. This change in mRNA specificity between Rps26-containing and deficient ribosomes arises from the recognition of the -4 position of the Kozak sequence by Rps26, thereby supporting the translation of well-translated mRNAs by Rps26-containing ribosomes in rich medium, and the translation of specific mRNAs in the Hog1 and Rim101 pathways by Rps26-deficient ribosomes that are formed under these stresses. What remains unknown is how Rps26-deficient ribosomes form under high salt and high pH stress. My preliminary results suggest Rps26-deficient ribosomes are produced by release of Rps26 from pre-existing ribosomes. Therefore, I will further dissect in more detail the mechanism that leads to the production of Rps26- deficient ribosomes, exploring the role of the Rps26-specific chaperone Tsr2 in delivering to and extracting Rps26 from ribosomes (Aim1), and testing which posttranslational modifications regulate this pathway (Aim2). Next, I will test if Rps26 is (re-) incorporated into Rps26-deficient ribosomes, to allow for a rapid switch in mRNA-specificity without the costs of re-building new ribosomes, or whether instead these ribosomes are degraded (Aim3). Together, these experiments will clarify how Rps26-deficient ribosomes form under stress. In addition to further expanding on this novel paradigm of stress-induced production of a specific ribosome population, the results will also have implications for the development of diseases linked to Rps26-deficiency, such as Diamond- Blackfan anemia. Because the etiology of 10-15% of all cases remains unknown, and is not linked to RP- deficiency, it is possible that overactivation of pathways leading to the release of RPs such as Rps26 might be responsible for a subset of cases, similar to the subset of cases caused by deficiency of the Rps26-chaperone Tsr2. Furthermore, ribosomes lacking individual RPs, including Rps26, are produced in cancer cells, where they are associated with poor outcomes. Thus, this work will also help delineate how cancer cells modulate the translational machinery to subvert translation to its purposes.